Opioid receptors are widely distributed in the central and peripheral nervous system and upon activation have manifold actions including; depression of respiration, activation of the reward pathway, disruption of normal gastrointestinal motility and analgesia. It is currently difficult to examine this widespread distribution in wild type animals. Antibodies against the opioid receptors are often very unreliable and the epitopes are most often intracellular such that detection in living tissue is not possible. The use of transgenic or knockin animals to identify receptors requires extensive characterization, can be limited by varied expression levels and has been limited to studies in mice. The goal of this proposal is to use a ligand directed approach to covalently label endogenous opioid receptors in living tissue. The guide compound, naltrexamine, is a non-selective opioid antagonist that has high affinity for all subtype opioid receptors. Naltrexamine will be coupled through a series of linkers, a reactive acyl imidazole and fluorescent ligand. Once bound to the receptor, the reactive acyl imidazole reacts with a lysine on the receptor and simultaneously releases the naltrexamine. Thus natrexamine is free to dissociate from the receptor, however the fluorescent ligand is left covalently bound to the receptor. Preliminary results indicate that this compound interacts very effectively with the mu-opioid receptor. Thus endogenous receptor are labeled and the labeling is not dependent on species. This compound and future refinements will be used areas of the CNS where opioid receptors are expressed in only a small population of neurons. This proposal will center on the VTA and Substantia Nigra in mouse and rat. Opioid receptor expressing neurons in living brain slices will be identified and whole cell recordings will be made. By targeting these cells a complete understanding of opioid action in this key area of the reward system will be possible. The results of this cell based study will address a long-standing question surrounding the mechanisms that underlie one aspect of the addictive properties of opioids.